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. 2022 Feb:414:108410.
doi: 10.1016/j.heares.2021.108410. Epub 2021 Dec 7.

Role of GluA4 in the acoustic and tactile startle responses

Sofía García-Hernández  1 , María E Rubio  2
Affiliations

Role of GluA4 in the acoustic and tactile startle responses

Sofía García-Hernández et al. Hear Res. 2022 Feb.

Abstract

The primary startle response (SR) is an innate reaction evoked by sudden and intense acoustic, tactile or visual stimuli. In rodents and humans the SR involves reflexive contractions of the face, neck and limb muscles. The acoustic startle response (ASR) pathway consists of auditory nerve fibers (AN), cochlear root neurons (CRNs) and giant neurons of the caudal pontine reticular nucleus (PnC), which synapse on cranial and spinal motor neurons. The tactile startle response (TSR) is transmitted by primary sensory neurons to the principal sensory (Pr5) and spinal (Sp5) trigeminal nuclei. The ventral part of Pr5 projects directly to the PnC neurons. The SR requires rapid transmission of sensory information to initiate a fast motor response. Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR) are necessary to transmit auditory information to the PnC neurons and elicit the SR. AMPARs containing the glutamate AMPAR subunit 4 (GluA4) have fast kinetics, which makes them ideal candidates to transmit the SR signal. This study examined the role of GluA4 within the primary SR pathway by using GluA4 knockout (GluA4-KO) mice. Deletion of GluA4 considerably decreased the amplitude and probability of successful ASR and TSR, indicating that the presence of this subunit is critical at a common station within the startle pathway. We conclude that deletion of GluA4 affects the transmission of sensory signals from acoustic and tactile pathways to the motor component of the startle reflex. Therefore, GluA4 is required for the full response and for reliable elicitation of the startle response.

Keywords: AMPAR; Caudal pontine reticular nucleus (PnC); GluA4; Principal sensory trigeminal nucleus (Pr5); Startle reflex.

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Figures

Figure 1.
Figure 1.. Diagram representing current knowledge of the acoustic and tactile startle pathways relevant to this study.
High intensity (broadband or octave centered) acoustic stimulation delivered suddenly activates hair cells in the cochlea, which send acoustic information through the spiral ganglion neurons that form the auditory nerve (AN). The AN sends signals to large neurons in the cochlear root (CRN). The CRNs project mainly contralaterally to giant neurons in the caudal pontine reticular formation (PnC), which mediate the acoustic startle response. Air puffs applied to the dorsum of the head effectively stimulate skin receptors that send tactile information through the trigeminal nerve, mainly the ophthalmic branch. The trigeminal nerve transmits the signal mainly to neurons in the principal sensory nucleus (Pr5). The caudal Pr5 projects directly to the PnC, which mediates the tactile startle response. Acoustic and tactile startle modalities converge on giant PnC neurons, which project directly to motoneurons and motor interneurons in the spinal cord. These spinal motoneurons in turn activate skeletal muscles to elicit a fast motor response.
Figure 2.
Figure 2.. Deletion of GluA4 reduces the acoustic startle response (ASR).
a, Representative ASR waveform from a WT (black) and a GluA4-KO (red) mouse, elicited with 115 dB of white noise for 40ms (horizontal gray bar); the vertical bar in the second peak indicates the maximum peak amplitude detected automatically by the system (plotted in b). b, Mean maximum 2nd peak amplitude (±SEM) of the ASR as a function of sound level (dB) is significantly decreased in KO as compared to WT mice (F(1,15)=102.6, p< 0.0001; RM two way-ANOVA; n= 7 WT, 10 KO; Cohen’s f=2.4). There was also a significant effect of sound level (F(2.2,32.8)=105.8, p< 0.0001; RM two way-ANOVA; n= 7 WT, 10 KO; Cohen’s f=2.7). The threshold for the ASR is higher in the GluA4KO than in the WT mice (arrows) as shown by the genotype x sound level interaction (F(5,75)= 59.4, p< 0.0001; RM two way-ANOVA; n= 7 WT, 10 KO; Cohen’s f=2.0). c-d, Example of waveform traces of the ASR from WT and GluA4-KO mice used to determine the amplitude of the 1st peak (arrowheads) and the percentage of successful trials. e, Mean percentage of successful trials of the ASR is significantly reduced in the KO as compared to WT mice (F(1,16) = 19.8, p= 0.0004; RM two way-ANOVA; n= 8 WT, 10 KO; Cohen’s f=1.2). There was no effect of sound level (F(1.97,31.6)=1.34; p=0.28) and no interaction between genotype x sound level (F(2,32)=0.67, p=0.52). f, Mean amplitude of the 1st peak in successful trials of the ASR is significantly reduced in the KO as compared to WT mice (F(1,16) = 54.5, p< 0.0001; RM two way-ANOVA; n= 8 WT, 10 KO; Cohen’s f=3.0). There was an effect of sound level (F(1.78,28.4)=18.4, p< 0.0001; RM two way-ANOVA; n= 8 WT, 10 KO; Cohen’s f=1.1), and a low genotype x sound level interaction (F(2,32)=4.9, p=0.01; Cohen’s f=0.5).
Figure 3.
Figure 3.. Lack of GluA4 does not affect auditory sensitivity.
a, Averaged ABR waveforms evoked with clicks from WT (black) and GluA4-KO (red) mice. b, Mean ABR thresholds (±SEM) are similar between genotypes (F(1,28) = 1.41, p= 0. 24; RM two way-ANOVA; n= 15 each genotype). There was an effect of stimulus frequency (F(2.8,78)=85.9, p< 0.0001; RM two way-ANOVA; n= 15 each genotype; Cohen’s f=1.7), and there was no genotype x frequency interaction (F(6,168)=0.59, p=0.74).c-d, Mean amplitude (±SEM) as a function of sound level (dB) for peaks 1 and 2 (P1 and P2) from click-evoked ABRs. Amplitudes of P1 and P2 are similar between genotypes (P1: F(1,26) = 0.13, p= 0.71; P2: F(1,26) = 0.20, p= 0.65; RM two way-ANOVA; n= 14 each genotype). There was an effect of sound level (P1: F(2.01,52.3)=139.5, p< 0.0001; RM two way-ANOVA; n= 14 each genotype; Cohen’s f=2.3; P2: F(2.01,52.3)=81, p< 0.0001; RM two way-ANOVA; n= 14 each genotype; Cohen’s f=2.3), and there was no genotype x sound level interaction (P1: F(3,78)=0.2, p=0.89; P2: F(3,78)=1.4, p=0.24).
Figure 4.
Figure 4.. Deletion of GluA4 reduces the tactile startle response (TSR).
a, Representative TSR waveform from a WT (black) and a GluA4-KO (red) mouse, elicited with air puffs. Such waveforms were used to determine the amplitude of the 1st peak (arrowhead) and the percentage of successful trials. b, Mean amplitude of the 1st peak in successful trials of the TSR is significantly reduced in KO (0.240 ± 0.08 SD) as compared to WT mice (0.86 ± 0.19 SD) (t (9.49)= 8.21, p<0.0001, unpaired t-test with Welch’s correction; n= 8 each genotype; Cohen’s d=4.1). c, Mean percentage of successful trials of the TSR is significantly reduced in the GluA4-KO (83.9 ± 10.4 SD) as compared to WT mice (97.8 ± 2.0 SD) (t (7.47)= 3.68, p=0.007, unpaired t-test with Welch’s correction; n= 9 WT, 8 KO; Cohen’s d=2.1).
Figure 5.
Figure 5.. Deletion of GluA4 does not affect the motor component of the SR.
a, Mean forelimb grip strength is similar between WT (6.7 ± 1.2 SD) and KO (6.3 ± 1.2 SD) mice (t (18)= 0.68, p= 0.51, unpaired t-test; n= 9 WT, 11 KO). b, Mean body weight is similar between genotypes (WT: 25.2 ± 2.5 SD; KO: 24.6 ± 1.9 SD) (t (60)= 1.0, p= 0.32, unpaired t-test; n= 30 WT, 32 KO). c, Mean peak ASR latency is similar between genotypes (WT: 17.77 ± 0.74 SD; KO:17.75 ± 1.5 SD) (t (7.12)= 0.02, p= 0. 98, unpaired t-test with Welch’s correction; n= 6 each genotype). d, Mean peak TSR latency is significantly increased in the GluA4-KO (31.6 ± 0.9 SD) as compared to WT mice (30.8 ± 0.3 SD) (t (12)= 2.39, p= 0. 03, unpaired t-test; n= 7 each genotype; Cohen’s d=1.3).
Figure 6.
Figure 6.. Lack of GluA4 affects short-term habituation of the ASR.
a, Mean maximum 1st peak amplitude (±SEM) of the ASR as a function of repeated acoustic stimulation (50 white noise pulses of 105 dB, 40 ms per pulse, with Inter-trial intervals of 20 s). b, To obtain the percentage change of the startle response (shown in b and e), the fifty trials from plots a and d, respectively, were split into blocks of five trials, and each block of five trials averaged for each mouse; the percentage of startle response in each block is relative to the first block. Mean percentage of the initial startle response is similar between genotypes (F(1,10)=3.5, p= 0.09; RM two way-ANOVA; n= 6 each genotype); there is a significant effect of block number (F(2.54,25.4)=3.49, p=0.03; RM two way-ANOVA; n= 6 each genotype; Cohen’s f=0.59); and there is no genotype x block interaction (F(9,90)=1.15, p=0.33; RM two way-ANOVA; n= 6 each genotype). c, To express habituation, the averaged amplitude of the last ten trials was expressed as a ratio to the averaged amplitude of the first five trials for each mouse, and the mean (±SEM) was plotted as a bar. Short-term habituation of the ASR is significantly increased in the GluA4-KO (0.61 ± 0.22 SD) as compared to WT (0.88 ± 0.10 SD) mice (t(10)=2.68, p= 0.02, unpaired t-test; n= 6 each genotype; Cohen’s d=1.6). d, Mean maximum 1st peak amplitude (±SEM) of the TSR as a function of repeated tactile stimulation (50 air puffs, 40 ms each, with Inter-trial intervals of 20s). e, Mean percentage of the initial startle response is similar between genotypes (F(1,12)=0.06, p= 0.8; RM two way-ANOVA; n= 7 each genotype); there is no significant effect of block number (F(2.77,33.3)=2.9, p=0.05); and there is no genotype x block interaction (F(9,108)=0.69, p=0.71; RM two way-ANOVA; n= 7 each genotype). f, Short-term habituation of the TSR (expressed as described in b above) is similar between genotypes (WT: 0.78 ± 0.25 SD; KO: 0.79 ± 0.29 SD) (t(12)=0.11, p= 0.91, unpaired t-test; n= 7 each genotype).
Figure 7.
Figure 7.. GluA4 is localized in the primary SR pathway.
a, Immunofluorescence of GluA4 is in the cerebellum (Cer) labeling the Bergmann glia (BG), the anteroventral cochlear nucleus (AVCN) and in the cochlear root nucleus (CRN). b, GluA4 is also localized in the caudal pontine reticular nucleus (PnC), the motor nucleus of the trigeminal nerve (Mo5), the principal sensory nucleus of the trigeminal nerve (Pr5) and the medial nucleus of the trapezoid body (MNTB). c, The cell bodies of PnC giant neurons (arrowheads) are immunolabeled with GluA4. d, GluA4 in the CRN is observed surrounding calbindin (calb) labeled cell bodies. e, GluA4 in the Pr5 is mainly in the neuropil and in some small cell bodies (arrow pointing to one cell); doted line divides the Pr5 into the dorsal (above) and the ventral (below) parts of the nucleus. Scale bars: a, 200 μm; b, 100 μm; c-e, 50 μm.
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